Biomanufacturing system, method, and 3D bioprinting hardware in a reduced gravity environment

ABSTRACT

A method, apparatus, and system are provided for the printing and maturation of living tissue in an Earth-referenced reduced gravity environment such as that found on a spacecraft or on other celestial bodies. The printing may be three-dimensional structures. The printed structures may be manufactured from low viscosity biomaterials.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part patent application of andclaims priority and benefit under 35 U.S.C. § 120 to copending U.S.patent application Ser. No. 15/225,547 filed on Aug. 1, 2016, whichclaims priority to U.S. Provisional App. No. 62/199,793, filed on Jul.31, 2015, the entire contents of all of the forgoing are incorporated byreference in their entirety.

BACKGROUND

The present embodiments relate to a system, method, and apparatus forbioprinting in a reduced gravity environment.

SUMMARY

In some embodiments, a method for the additive manufacturing of livingtissue in a reduced gravity environment may comprise one or more of thesteps of providing a reduced gravity environment, providing a housinghaving a bioprinter, providing one or more bioinks, and printing one ormore three-dimensional tissues with the one or more bioinks from saidbioprinter within the reduced gravity environment. One or morethree-dimensional tissues may be printed in a bioreactor. In use, thebioreactor may be positioned on at least one print stage of thebioprinter. Another step may include positioning the one or morethree-dimensional tissues into a bioreactor after printing. The one ormore three-dimensional tissues may be positioned into the bioreactormanually or automatically. Further one or more bioinks may includenon-living biological components such as at least one of natural orsynthetic structural proteins, polymers, macromolecules, orpharmaceuticals. The reduced gravity environment may be an environmentwherein the gravitational acceleration is less than 9.807 meters persecond per second. In addition, one or more bioinks may include livingbiological components such as at least one of undifferentiated stemcells, partially differentiated stem cells, terminally differentiatedcells, microvascular fragments, or organelles.

In addition, in some embodiments, the method may include the step ofmaturing one or more three-dimensional tissues. The one or more bioinksmay have a viscosity range of approximately 1 to 10,000,000 centipoise,preferably the viscosity range is approximately 5 to 2,000 centipoise.Another step may include controlling at least one of temperature orhumidity. Further steps may include providing one or more print stagesand controlling the thermal characteristics of the one or more printstages.

In some embodiments, further steps may provide one or more print headsand controlling the thermal characteristics of the one or more printheads. There may be additional steps of controlling the temperature ofone or more bioinks. Additional steps of at least partially controllingthe additive manufacturing of living tissue in the reduced gravityenvironment from one or more locations may be used. The one location maybe terrestrial. The one or more three-dimensional tissues may betransported from the reduced gravity environment to a different gravityenvironment. The different gravity environment may be at least one of aterrestrial environment or an extraterrestrial environment. Anotherembodiment may include the step of incorporating prefabricated structureinto one or more three dimensional tissues for at least one of thecreation of the tissue or organ, support structure, perfusion aid,implantation aid, cell delivery, or reagent delivery.

In some embodiments, a biomanufacturing system capable of assembling andmaturing living tissue in a reduced gravity environment from one or morebioinks may include a bioprinter, a cell culturing device, one or morebioinks, and an environment of reduced gravity surrounding thebioprinter and the cell culturing device. The bioprinter may be a threedimensional printer. Further the bioprinter may be separate from thecell culturing device. The cell culturing device may include at leastone of a mechanical tissue stimulation or electrical tissue stimulation.

In addition, in some embodiments, one or more print heads of thebioprinter may be in fluid communication with the interior of the cellculturing device. Further the environment of reduced gravity may betemperature controlled and/or humidity controlled. In addition, one ormore bioinks may have a viscosity range of approximately 1 to 10,000,000centipoise, preferably the viscosity range is approximately 5 to 2,000centipoise. The environment of reduced gravity may have a gravitationalacceleration less than 9.807 meters per second per second. Further insome embodiments, at least one of the cell culturing device or the oneor more bioinks downstream of the bioprinter may be transported from theenvironment of reduced gravity to an environment having a differentgravity. In some embodiments, the system is a modular configuration. Themodular configuration may include both major systems and some individualcomponents that may be swapped-out for resupply, refurbishment, orupgrade. The modular configuration may include one or more of captivefasteners, self-aligning blind-mate electrical and mechanicalconnectors, grouping of low mean time between failure (MTBF) and highMTBF components, grouping of certain electrical components withinelectromagnetic interference shielding, and/or colocation of elementsrequiring air or liquid cooling. In addition, the cell culturing devicemay include an integrated life support system for transportation ofliving tissue from said environment of reduced gravity to an environmenthaving a different gravity.

Further, in some embodiments, an additive manufacturing apparatus mayinclude a reduced gravity environment, a bioprinter positioned in thereduced gravity environment such that the bioprinter has one or moreprint heads in relation to at least one print stage, and one or morebioinks have a viscosity range of approximately 1 to 10,000,000centipoise in fluid communication with the one or more print heads. Thebioprinter may be a three-dimensional printer.

In addition, in some embodiments, the apparatus may have a bioinkdispensing system, a visualization system capable of observing a topsurface of the print stage, an x-axis translation system, a y-axistranslation system, and a z-axis translation system, and wherein atleast one of the one or more print heads allow direct write constantpressure extrusion. Further embodiments may include one or morebioreactors. The viscosity range may be approximately 5 to 2,000centipoise. In addition, one or more thermoplastics may be in fluidcommunication with the one or more print heads. Further the housing mayhave at least one of temperature control or humidity control. In someembodiments the apparatus is a modular configuration. The modularconfiguration may include both major systems and some individualcomponents that may be swapped-out for resupply, refurbishment, orupgrade. The modular configuration may include one or more of captivefasteners, self-aligning blind-mate electrical and mechanicalconnectors, grouping of low mean time between failure (MTBF) and highMTBF components, grouping of certain electrical components withinelectromagnetic interference shielding, and/or colocation of elementsrequiring air or liquid cooling.

In some embodiments, a bioreactor for receiving a printed tissue maycomprise a housing defining a volume therein. In various embodiments,the bioreactor may include a print platform within the volume of thehousing. In some embodiments, the bioreactor may include an intakemanifold and an outlet manifold positioned in the volume of the housing.In various embodiments, each one of the intake manifold and the outletmanifold includes one or more ports adapted to be in fluid communicationwith a printed tissue within the housing.

In addition, in some embodiments, at least one of the intake manifoldand the outlet manifold may include an electrical stimulation device. Invarious embodiments, the electrical stimulation device may include aconductive material positioned on one or more surfaces of the at leastone of the intake manifold and the outlet manifold. In some embodiments,the conductive material may include one or more needles projecting andadapted to engage a printed tissue. In various embodiments, at least oneof the intake manifold and the outlet manifold may include one or moreneedles defining the one or more ports. Moreover, in some embodiments,at least one of the intake manifold and the outlet manifold may includeone or more channels in communication with the one or more ports. Invarious embodiments, at least one of the intake manifold and the outletmanifold may be moveable relative to each other. In some embodiments,the bioreactor may include a drive mechanism moving at least one of theintake manifold and the outlet manifold. In various embodiments, thebioreactor may include one or more air traps in fluid communication withthe one or more ports. In some embodiments, the bioreactor may includeone or more detachable feed bags and waste bags.

In various embodiments, a bioreactor for receiving a printed tissue maycomprise a housing having one or more manifolds therein adapted toengage a printed tissue. In some embodiments, the one or more manifoldsmay have a plurality of perfusion ports in fluid communication a printedtissue. In various embodiments, the one or more manifolds may have oneor more electrical stimulation devices. In some embodiments, the one ormore manifolds may have a mechanical stimulation device connectedthereto.

In addition, in various embodiments, at least one of the one or moremanifolds may have one of the plurality of perfusion ports, the one ormore electrical stimulation devices, and the mechanical stimulationdevice. In some embodiments, the bioreactor may include a printplatform. In various embodiments, at least one of the one or moremanifolds may contain the plurality of perfusion ports defined by one ormore needles. In various embodiments, the mechanical stimulation devicemay move the one or more manifolds between a compressed position and anextended position. Moreover, in some embodiments, the one or moreelectrical stimulation devices may be an exterior coating to the one ormore manifolds. In various embodiments, the exterior coating may includeone or more needles. In some embodiments, the one or more manifolds mayinclude an intake manifold and an outlet manifold.

In some embodiments, a method of culturing a printed tissue in abioreactor in a reduced gravity environment may comprise the step ofproviding a reduced gravity environment. In various embodiments, themethod may include providing one or more bioreactors in the reducedgravity environment. In some embodiments, the method may includeprinting one or more tissues into the one or more bioreactors. Invarious embodiments, the method may include providing one or moreperfusions to one or more printed tissues. In some embodiments, themethod may include providing one or more electrical stimulations to oneor more printed tissues. Moreover, in some embodiments, the method mayinclude providing one or more mechanical stimulations to one or moreprinted tissues.

In addition, in some embodiments, the method may include removing airfrom fluid communication within the one or more bioreactors. In variousembodiments, the method may include engaging one or more manifolds withthe one or more printed tissues. In some embodiments, the method mayinclude fluidly engaging one or more of at least one of a feed bag and awaste bag to the one or more bioreactors. In various embodiments, themethod of providing one or more perfusions to one or more printedtissues may include the step of passing fluid from an intake manifoldthrough the one or more printed tissues to an outlet manifold. Moreover,in some embodiments, the method may include recirculating fluid betweenthe intake manifold, the one or more printed tissues, and the outletmanifold.

In various embodiments, a method of culturing a printed tissue in abioreactor in a reduced gravity environment may comprise the steps ofproviding a reduced gravity environment. In some embodiments, the methodmay include providing one or more bioreactors in the reduced gravityenvironment. In various embodiments, the method may include printing oneor more tissues into the one or more bioreactors. In some embodiments,the method may include providing an intake manifold and an outletmanifold, each one of the intake manifold and the outlet manifold havingone or more ports. In various embodiments, the method may includeproviding one or more perfusions to one or more printed tissues throughthe one or more ports of the intake manifold. In some embodiments, themethod may include providing one or more electrical stimulations to oneor more printed tissues by contact with at least one of the intakemanifold and the outlet manifold. Moreover, in various embodiments, themethod may include providing one or more mechanical stimulations to oneor more printed tissues by moving at least one of the intake manifoldand the outlet manifold.

In addition, in various embodiments, the method of providing one or moremechanical stimulations may include at least one of compression orextending by moving at least one of the intake manifold and the outletmanifold. In some embodiments, the method may include engaging at leastone of the intake manifolds and the outlet manifold with the one or moreprinted tissues. In various embodiments, the method may include fluidlyengaging the one or more ports of the intake manifold and the outletmanifold with one or more vessels within the one or more printedtissues. In some embodiments, the method of providing one or moreperfusions to the one or more printed tissues may include a plurality ofthe one or more ports defined by a plurality of needles. In variousembodiments, the method may include removing air from fluidcommunication within a housing within the one or more bioreactors.Moreover, in some embodiments, the method of printing one or moretissues into the one or more bioreactors may include the step ofremoving the one or more bioreactors from a 3D printer.

In some embodiments, a bioreactor for receiving a printed tissue maycomprise a housing defining a volume therein. In various embodiments,the bioreactor may include a print platform within the volume of thehousing. In some embodiments, the bioreactor my include an intakemanifold and an outlet manifold positioned in the volume of the housing.In various embodiments, each one of the intake manifold and the outletmanifold may include one or more ports. Moreover, in some embodiments,the bioreactor may include at least one first air trap in fluidcommunication with the one or more ports of at least one of the intakemanifold and the outlet manifold.

In addition, in some embodiments, at least one of the intake manifoldand the outlet manifold includes an electrical stimulation device. Invarious embodiments, the bioreactor may include a second air trap. Insome embodiments, the second air trap may be in fluid communication withthe volume of the housing and a feed bag. In various embodiments, thebioreactor may include a waste bag in fluid communication with thevolume of the housing. In some embodiments, the bioreactor may include apump in fluid communication with the at least one first air trap. Invarious embodiments, at least one of the intake manifold and the outletmanifold may be moveable relative to each other.

In various embodiments, a bioreactor for receiving a printed tissue maycomprise a housing having a volume, wherein the housing has one or moremanifolds therein adapted to engage a printed tissue. In someembodiments, the one or more manifolds may have a plurality of perfusionports. In various embodiments, the one or more manifolds may have one ormore electrical stimulation devices. In some embodiments, the one ormore manifolds may have a mechanical stimulation device connectedthereto. In various embodiments, the bioreactor may include one or moreair traps in fluid communication with the volume defined by the housing.

In addition, in various embodiments, at least one air trap may be influid communication with at least one of the plurality of perfusionports of the one or more manifolds within the housing. In someembodiments, at least one of the one or more manifolds may have one ofthe plurality of perfusion ports, the one or more electrical stimulationdevices, and the mechanical stimulation device. In various embodiments,the one or more manifolds may include an intake manifold and an outletmanifold. In some embodiments, one or more air traps may be in fluidcommunication with each one of the intake manifold and the outletmanifold. In various embodiments, one or more air traps may be in fluidcommunication with the volume of the housing and a feed bag. In someembodiments, at least one of the one or more manifolds may contain theplurality of perfusion ports defined by one or more needles. Moreover,in some embodiments, the mechanical stimulation device may move the oneor more manifolds between a compressed position and an extendedposition.

In some embodiments, a method of culturing a printed tissue in abioreactor in a reduced gravity environment may comprise the steps ofproviding a reduced gravity environment. In various embodiments, themethod may include providing one or more bioreactors in the reducedgravity environment. In some embodiments, the method may includeprinting one or more tissues into the one or more bioreactors. Invarious embodiments, the method may include providing one or moreperfusions to one or more printed tissues. Moreover, in someembodiments, the method may include removing air from the one or morebioreactors.

In addition, in some embodiments, the method may include one or more airtraps in fluid communication with one or more manifolds. In variousembodiments, the method of providing one or more perfusions to one ormore printed tissues may include the step of recirculating fluid withinthe one or more bioreactors. In some embodiments, the method of removingair from the one or more bioreactors may occur during the step ofrecirculating fluid within the one or more bioreactors. In variousembodiments, the method of removing air from the one or more bioreactorsmay include the step of priming one or more ports of one or moremanifolds. In some embodiments, the method may include engaging the oneor more manifolds to the printed tissue. In various embodiments, themethod of removing air from the one or more bioreactors may includeevacuating air from a volume defined by a housing of at least one of theone or more bioreactors. Moreover, in some embodiments, the method mayinclude at least one of the steps of providing one or more electricalstimulations to the one or more printed tissues and providing one ormore mechanical stimulations to the one or more printed tissues.

In various embodiments, a method of culturing a printed tissue in abioreactor in a reduced gravity environment may comprise the steps ofproviding a reduced gravity environment. In some embodiments, the methodmay include providing a printed tissue in a housing of a bioreactor inthe reduced gravity environment. In some embodiments, the method mayinclude recirculating fluid to the printed tissue. Moreover, in variousembodiments, the method may include removing air from the fluidrecirculating to the printed tissue.

In addition, in various embodiments, the method may include supplyingfluid from a feed bag to the housing. In some embodiments, the methodmay include removing air from the fluid communication of the feed bag tothe housing. In various embodiments, the method may include priming oneor more manifolds within the bioreactor with fluid. In variousembodiments, the method may include engaging one or more manifolds withthe printed tissue. In some embodiments, the method of recirculatingfluid to the printed tissue may include recirculating fluid into one ormore manifolds. In various embodiments, the method may includeevacuating air from the housing. In some embodiments, the method mayinclude printing the printed tissue in the housing of the bioreactor. Invarious embodiments, the method may include sealing the housing afterthe step of printing the printed tissue in the housing of thebioreactor. In some embodiments, the method may include at least one ofthe step of removing the bioreactor from a 3D bioprinter and printing asecond printed tissue in a second bioreactor. Moreover, in someembodiments, the method may include at least one of the steps ofproviding one or more electrical stimulations to the printed tissue andproviding one or more mechanical stimulations to the printed tissue.

These and other advantages and features, which characterize theembodiments, are set forth in the claims annexed hereto and form afurther part hereof. However, for a better understanding of theembodiments, and of the advantages and objectives attained through itsuse, reference should be made to the Drawings, and to the accompanyingdescriptive matter, in which there is described example embodiments.This summary is merely provided to introduce a selection of conceptsthat are further described below in the detailed description, and is notintended to identify key or essential features of the claimed subjectmatter, nor is it intended to be used as an aid in limiting the scope ofthe claimed subject matter.

BRIEF DESCRIPTION OF THE FIGURES

In the drawings, like reference characters generally referred to thesame parts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating the principles of the invention.

FIG. 1 illustrates an embodiment of the biomanufacturing system whereinthe cell culturing bioreactor and 3D bioprinter are combined in a singleintegrated biomanufacturing facility capable of manufacturing tissue inreduced gravity, showing the bioprinter and the bioreactor outside theenclosure for clarity.

FIG. 2 is a perspective view of a prior art heart ventricle influencedby the negative effects of gravity; the material of the 3D printedstructure flowed out of the desired geometry due to gravity.

FIG. 3 is a perspective view of the integrated reduced gravitybiomanufacturing facility of FIG. 1 with the door closed.

FIG. 4 is a perspective view depicting the general layout of some of theexterior biomanufacturing facility components with the door open.

FIG. 5 is a perspective view of the reduced gravity 3D bioprinter printstage and interdependent systems that translate the stage and feed thebioinks to the print heads.

FIG. 6 is a perspective view of prior art terrestrial electrospinninghardware used to make prefabricated structures that can be incorporatedinto bioprinted tissues.

FIG. 7 is a perspective view of an embodiment of the print stageincluding a flexible bioreactor capable of mechanical and electricalstimulation of a printed tissue.

FIG. 8A is an enlarged side view of a portion of a prior art additivestructure influenced by the negative effects of gravity.

FIG. 8B is a sectional view of the prior art additive structure of FIG.8A influenced by the negative effects of gravity.

FIG. 9A is an enlarged side view of a portion of an embodiment of theadditive structure printed in a reduced gravity environment.

FIG. 9B is a sectional view of the embodiment of the additive structureof FIG. 9A.

FIG. 10 is a perspective view of another embodiment of a bioreactor withthe cassette lid exploded therefrom.

FIG. 11 is a perspective view of the bioreactor of FIG. 10 rotated 180degrees with the cassette, locking mechanism, housing lid removedillustrating an embodiment of the manifolds disengaged from thebioprinted construct.

FIG. 12 is a perspective exploded view of the bioreactor of FIG. 10 .

FIG. 13 is a perspective view of an embodiment of an intake manifold ofthe bioreactor of FIG. 10 .

FIG. 14 is another perspective view of the intake manifold of FIG. 13with the electrical stimulation device exploded therefrom.

FIG. 14 a is a perspective view of the flow channels through the intakemanifold of FIGS. 13 and 14 .

FIG. 15 is a perspective view of an embodiment of an outlet manifold ofthe bioreactor of FIG. 10 with the electrical stimulation deviceexploded therefrom.

FIG. 16 is another perspective view of the outlet manifold of FIG. 15 .

FIG. 17 is a schematic view of one embodiment of the fluid flow of thebioreactor of FIG. 10 .

DETAILED DESCRIPTION

Various embodiments of the invention may include a biomanufacturingsystem, method, and 3D bioprinting hardware optimized for exclusive usein a reduced gravity environment 30 such as that found on an orbitingspacecraft (microgravity) or another celestial body (fractionalgravity). FIG. 1 illustrates a cell culturing device or bioreactor 3, a3D bioprinter 2, and/or integrated biomanufacturing facility 1 capableof manufacturing tissue in reduced gravity. In the partial or completeabsence of gravity, this system is able to construct tissues usingbioinks with lower viscosities than are currently feasible for allEarth-based bioprinters. Lower viscosities allow faster printing withoutdamaging the cells, proteins, and biomacromolecules from the effects ofcavitation, high pressure, or chemical crosslinking agents. Anotheradvantage of lower viscosity is cell motility within the printed tissue.This allows the tissues to mature faster and reduce hindrance in theformation of vascular beds used for thick tissue viability. FIG. 2illustrates a prior art heart ventricle 4 influenced by the negativeeffects of gravity, resulting in deformity out of the desired shape.This deformity out of the desired pattern may result in improperfunction.

In addition to reduced viscosity, tissues can be built in thebiomanufacturing facility 1 without or with reduced external supportstructures. In traditional, terrestrial additive manufacturing,overhangs may be supported either with the same material or a separatematerial. In either case, this material is removed after processing asit is not part of the desired tissue or end product; it is merely ameans to perform bottom-up construction. In the absence of a pronouncedgravitational vector, such as in the environment in Low Earth Orbit(LEO), these tissues can be built with only the functional components.This reduces the risk that supports may be forgotten and left in atissue, or left out and have an inner passage form incompletely.Additionally, more complex geometries can be produced containingenclosed void volumes, such as the four chambers of a heart, that areunattainable using similar systems on Earth.

Finally, basic stem cell research on the International Space Station(ISS) has demonstrated improved proliferation, maturation, anddifferentiation. Expanding upon these findings during the culture phaseof this system can produce more robust tissues and produce those tissuesfaster and more easily. This allows the system to use lower cellconcentrations and culture for shorter periods of time than Earth-basedsystems to produce the same or superior tissue. For complex tissues,this time savings could be substantial. Therefore, overall both thequality and quantity of the bio tissue may be dramatically improved inreduced gravity.

The reduced gravity biomanufacturing facility 1 comprising a 3Dbioprinter 2 and a cell culturing bioreactor 3, is designed tomanufacture 3D living tissue in a reduced gravity environment 30 such asthe microgravity environment of an orbiting spacecraft or the fractionalgravity environment on the surface of other celestial bodies such asEarth's moon (1.622 meters/second/second or about one-sixth Earth'sgravity) or Mars (3.711 meters/second/second or about one-third Earth'sgravity). For reference, the surface of planet Earth is considered tohave a unit gravity, or “1-g” environment equivalent to 9.807meters/second/second. Microgravity is a term often used to describe theweightless conditions experienced aboard a vehicle in a state ofcontinuous free fall as, for example, on a spacecraft in orbit around aplanet. A reduced gravity environment, therefore, is any environmentwith a gravitational acceleration less than that of the Earthenvironment. The physical effects of a reduced gravity environment are akey component of successful biomanufacturing. Since the biomanufacturingequipment uses a human habitable environment (atmosphere, thermal) forliving tissue, a variety of applications of, but is not limited to,microgravity spaceflight platforms may include International SpaceStation, commercial space stations such as the Bigelow Aerospace B330,or free-flyers such as the Space Exploration Technologies Corporation(SpaceX) DragonLab, Boeing CST-100 Starliner, or Sierra NevadaCorporation Dream Chaser®. Fractional gravity platforms might includerotating spacecraft or habitable facilities on or beneath the surface ofthe moon, Mars, an asteroid, or other extraterrestrial celestial bodies.

With reference to the prior art 4-layered printed wall 40 of FIGS. 8Aand 8B representing printing on Earth and a similarly constructed4-layered printed wall 50 of FIGS. 9A and 9B representing printing inreduced gravity, several advantages to reducing or removing gravity fromthe process are evident. Sedimentation within fluids does not occur.Under the influence of gravity, cells 41 sediment down to the ‘bottom’of each printed bead 42. Even after subsequent proliferation, thisinitially heterogeneous distribution of cells can result in structurallyweaker, less densely populated ‘top’ portions of each printed bead 42.Buoyance-driven stratification can also occur within the bulk materialof each bead 42. Conversely, the cells 41 and bulk material of printedwall 50 are naturally homogeneous in their positions throughout. Lowerviscosity fluids can be formed into 3D printed structures that stillmaintain their desired shapes without the complication ofgravity-induced deformation. The use of lower viscosities allows fasterprinting without damaging the cells, proteins, and biomacromoleculesfrom the effects of cavitation, high pressure, or chemical crosslinkingagents.

Another advantage of lower viscosity is cell motility within the printedtissue and cell interaction both within a bead of printed material andacross the boundary between beads of printed material. This allows thetissues to mature faster and reduces hindrance to the formation ofvascular beds used for thick tissue viability. Again with reference toFIGS. 8A, 8B, 9A, and 9B, prior art FIGS. 8A and 8B show the geometryresulting from the use of high viscosity fluids to create prior artprinted wall 40. Individual beads 42 virtually retain their circularcross section resulting in steep contact angles between successive beads42 and a relatively small contact interface 43. High viscosity fluidswill adhere to one another, but do not readily meld or intermix, so thearea of the contact interface 43 can act as a physical barrier to bothmass transport and cellular interaction between adjacent beads 42.Delamination between successive beads 42 can also occur due to therelatively small contact interface 43 and hindered ability for cells tointerconnect across this interface. Conversely, and advantageously, thelow viscosity beads 52 of printed structure 50 readily meld, the contactangles between adjacent beads 52 approach zero degrees as the material‘self levels’ along its height due to surface tension, and contact areas53 between adjacent beads 52 all but disappear. The resulting printedstructure 50 has a much more uniform overall width, cell andextracellular material distribution, and integrity.

In a reduced gravity environment, 3D bioprinter 2 can print using lowviscosity extrudable materials, hereafter referred to as bioinks, thatmay have one or more of the following components: natural and syntheticstructural proteins, such as fibrinogen, albumin, fibronectin, collagen,or hyaluronic acid; polymers, such as pluronic or urethanes; livingbiological components, such as undifferentiated stem cells, partiallydifferentiated stem cells, terminally differentiated cells,microvascular fragments, or organelles; macromolecules; orpharmaceuticals. These bioinks may have a viscosity as low asapproximately 1 centipoise on the low side, and have viscosities on thehigh side of typical bioinks used in terrestrial applications (forexample, on the order of 10,000,000 centipoise). Preferably the range ofviscosity is approximately 5 to 2,000 centipoise. On Earth, under theinfluence of gravity, structures printed using such low viscositybioinks cannot maintain their initial shape and will deform orstructurally fail under their own weight. Internal and/or externalscaffolds of like or dissimilar materials may be constructed to maintainthe initial shape. These scaffolds are subsequently removed. However, ina reduced gravity or near-weightless environment, complex shapes such ascantilevered overhangs and enclosed voids, such as the enclosed chambersof a heart, can be easily maintained. Yet another advantage of printingin a reduced gravity environment is the ability to build up cantileveredoverhangs that simply cannot be made on Earth, even with supportingscaffolds. On Earth, each new extruded bead typically contacts 75% ormore along the length of the bead directly beneath it. Therefore itutilizes many stacked but only slightly offset layers to incrementallybuild a cantilevered structure. In a reduced gravity environment, thegoal is to create structures wherein less than 50% of a new extrudedbead makes contact along the length of the bead directly beneath it.This will enable thinner overall printed structures and steeplycantilevered geometries that simply cannot be made on Earth using anyconventional means.

FIG. 2 demonstrates the root cause of further benefits of printing inreduced gravity using low-viscosity bioinks. In the additivemanufacturing process of bioprinting, material is extruded in successivelayers. The high viscosity bioinks used to terrestrially print theventricle 4 shown in FIG. 2 maintain clearly distinct layers on thefinal structure. The boundaries between successive layers act as bothphysical and chemical barriers to cell proliferation. Cells canrelatively easily interact within a single layer, but not across layerboundaries. The use of low viscosity bioinks advantageously results inlittle or no clearly defined layers in the resulting 3D printedstructure, thereby promoting and accelerating the interaction betweencells in different layers and leading to a more robust final product.

The reduced gravity biomanufacturing system comprises, at a minimum, twomajor subsystems: a 3D bioprinter and a cell culturing bioreactor inwhich a printed structure is incubated to promote cell proliferation,differentiation, and remodeling into a final product tissue. In oneembodiment, both subsystems occupy discrete and separate facilities.Precautions may be taken to prevent physical damage or exposure todeleterious environmental microorganisms during transfer betweensubsystems. In a preferred embodiment, both subsystems are containedwithin a single integrated facility. This embodiment reduces oreliminates moving a structure from bioprinter to bioreactor and protectsthe biomaterial during all stages of processing. FIGS. 3 and 4 show anembodiment of the reduced gravity biomanufacturing system whereby both3D bioprinter 2 and bioreactor 3 are housed in one integratedbiomanufacturing facility 1. Biomanufacturing facility 1 is a modularbox-like volume with an overall geometry compatible with the genericinterface requirements for various spacecraft or habitable systems.External enclosure or housing 5 includes a door 6 for operator access tothe environmentally controlled interior chamber where 3D bioprinter 2and cell culturing bioreactor 3 (not shown) reside. External enclosure 5surrounds and supports the individual assemblies and components withinthe unit and has exterior dimensions, in the embodiments shown, that areapproximately 21 inches×21 inches×18 inches (essentially a spaceflightdouble locker typical of the art). Several notional switches 11 enableoverall power to major subsystems. Circuit breaker 10 provides facilityovercurrent protection consistent with the requirements levied by thehabitable platform. Power connector 9 and data connector 8 are typicalmulti-pin shrouded connectors typical in the art for providingelectrical interfaces to a vehicle or facility. Consumables bays 7 housestock materials that are consumed by the 3D bioprinter 2 or cellculturing bioreactor 3 major subsystems. Consumables may include one ormore of bioinks used to print 3D structures, media to perfuse a 3Dprinted structure during cell proliferation, thermoplastic feed stockthat can be used to manufacture in situ bioreactor enclosures, andcompressed gas supplies such as oxygen used to maintain culturing cells.The hardware is designed in a modular configuration so that both majorsystems and some individual components can easily be swapped-out onorbit for resupply, refurbishment, or upgrade as technology advances.Modularity is further facilitated by design features such as the use ofcaptive fasteners that cannot be lost during removal in reduced gravity;self-aligning blind-mate electrical and mechanical connectors betweenmodular subsystems; logical grouping of low mean time between failure(MTBF) and high MTBF components separately to minimize the mass andvolume of replacements; grouping of certain electrical components withinelectromagnetic interference shielding; and colocation of elementsrequiring air or liquid cooling such as power supplies, thermoelectricPeltier devices.

A computerized command and data management system (CDMS)/power supply 23provides power, monitors, and controls operation of the facility 1. Theelectrical system components and topology are typical of those in theart of manufacturing high reliability, high safety equipment for themedical, defense, or aerospace fields. For example, the presentinvention uses a federated control architecture to reduce the risk ofmajor system failure resulting from the radiation and high-energyparticles often encountered in reduced gravity environments such asspace. (CDMS)/power supply 23 conditions the power and provides thevoltage levels used by the biomanufacturing facility as well asproviding electromagnetic interference filtering and electrical bonding.Software employed internally to operate and control components such aspumps, sensors, motors, and data acquisition are typical for computercontrolled electromechanical systems. Facility 1 has the ability tomonitor and control all of the system parameters real time with theadded flexibility of being able to uplink and downlink files, video, andoperating data at any time. The facility uses software and physicalinterfaces to various host vehicles or platforms that are compatiblewith command and control interfaces typical in the art such as universalserial bus (USB) and Ethernet. It may incorporate a digital display witha user-friendly graphical user interface (GUI). CDMS/power supply 23 maybe housed within enclosure 5 or may be a separate entity (not shown)connected via cables (not shown) to respective data connector 8 andpower connector 9. In a general sense, tasks performed by thebiomanufacturing system may be performed manually by an operator,semi-autonomously, or fully autonomously with or without remotemonitoring. For example, the remote monitoring may be at least partiallyfrom a terrestrial location. Biomanufacturing facility 1 may beergonomically designed to facilitate ease of use by an operator in areduced gravity environment.

FIG. 5 describes the reduced gravity 3D bioprinter 2 in greater detail.One or more print stages 12 comprises a flat plate that can be thermallycontrolled as desired. This plate may be metallic or nonmetallic,surface treated or untreated, removable or non-removable. In a preferredembodiment, print stage 12 is mounted to x-axis support structure 13 andy-axis support structure 14. Print stage 12 translates in two axesutilizing X-motion control system 25 and Y-motion control system 27 tomove in the X-direction and Y-direction respectively. In the preferredembodiment, control systems 25 and 27 comprise computer controlledbrushless DC servo motors common in the art to control the accuracy,repeatability, resolution, and velocity of print stage 12 during thebioprinting process. Support structures 13 and 14 are mounted withinenclosure 5 via vibration isolators 18 to further facilitate precisionin the printing process. Z-axis support structure 15 is mounted on aninterior sidewall of enclosure 5 directly above print stage 12. Eachprint head 17 includes an associated visualization system 24 anddispensing system 16 that will be described hereafter in greater detail.In a preferred embodiment, one or more print heads 17 each includeindividual Z-motion control systems 26 capable of independentlytranslating print heads 17 in a third, or Z-axis that is substantiallyorthogonal to the x-y plane defined by print stage 12. For example,control system 26 may comprise one or more computer controlled brushlessDC servo motors to control the accuracy, repeatability, resolution, andvelocity of one or more print heads 17 during the bioprinting process.In an enhanced embodiment, additional means for Z-axis translation ofdispensing system 16, and all print heads 17 may be desirable tofacilitate post-printing access to print stage 12. Using one or moretranslation or motion control systems such as but not limited to 25, 26,and 27, the relative position of print stage 12 and interdependentsystems can translate in the x-, y-, and z-directions up to about 12inches and feed stock to the print heads in the embodiment shown. In asecond embodiment, print stage 12 and, optionally, visualization system24, may translate in the z-direction while print heads 17 and dispensingsystem 16 may translate in the x- and y-directions. In a third alternateembodiment, print stage 12 may remain in a fixed location while z-axissupport structure 15 and dependent elements may translate in the x-, y-,and z-directions. In a fourth alternate embodiment, z-axis supportstructure 15 and dependent elements remain fixed while print stage 12may translate in the x-, y-, and z-directions. Each of the threealternate embodiments utilize different associations with motion controlsystems 25, 26, and 27 than shown in the preferred embodiment of FIG. 5. While weightless, or nearly weightless, the printed structure stillexperiences momentum. Abrupt changes in direction of the printedstructure may result in deformity. In some combinations of translationinterdependency described above, the printed structure remainsstationary or moves minimally in the x-direction, particularly thosewherein print heads 17 translate in the x- and y-directions. Incombinations wherein print stage 12, and hence the printed structure,translate in the x- and y-directions, both translation acceleration andvelocity are carefully controlled to mitigate momentum effects.

Working in concert with print stage 12 is multi-solution dispensingsystem 16 that incorporates precision control of the feed rates of thebioink fluids delivered to one or more removable and replaceable printheads 17. Being a dynamic system capable of 6-axes of freedom,dispensing system 16 is also able to maintain dynamic flow controlduring the bioprinting process all within a thermally controlledenvironment. Dispensing system 16 also provides precise start andendpoint volumetric control. Print technologies comprise two groups:point by point “ink jet” printer-based, also called laserjet printing,or point and line “direct write” syringe-based. In a direct writesystem, pressure is maintained, either mechanically (linear motor, drivescrew) or pneumatically (vacuum, pressurized gas, pressurized drivefluid), on a reservoir of bioink that is ejected through a small gaugeneedle or extruder tip to the printing substrate which is oftentemperature controlled. Feed is enabled by control of a valve (notshown). The feed rate and the ability to start and stop the flow ofmaterial differentiate the systems as well as the ability to handle awide range of working fluid viscosities. The preferred embodimentutilizes direct write print heads such as the SmartPump™ manufactured bynScrypt, Inc. (Orlando, Fla.) and may use either a very fine needle or avery fine ceramic tip extruder. The diameter of the extruded material istypically in the range of 12.5 to 125 micrometers. A plurality of printheads can be simultaneously or serially orchestrated to incorporateseveral different bio-inks into the printed structure. This feature mayallow the production of complex structures such as organs that mayutilize several different functional tissue types. As described below,some print heads may print non-biological material such asthermoplastics to build in situ bioreactor vessels or electricallyconductive material to electrically connect prefabricated sensors thatcan be incorporated into the 3D printed tissue. Surfaces of print heads17 and/or print stage 12 may be natively, or treated to be, hydrophobicor hydrophilic in order to urge the proper behavior of the extrudedbioinks. Physical forces such as surface tension are known to play amore dominant role in reduced gravity fluid dynamics.

Illuminated visualization system 24 may have one or more small cameras,associated with each print head 17, focused on print stage 12 pluseither visible or infrared illumination as is typical in the art such asLED lighting. Visualization system 24 may incorporate the ability tocapture both still and video images of the entire bioprinting process.The frame rate, resolution, and field of view are all fullyprogrammable. Illumination can be turned on or off by an operator asdesired. Observation of the structure during printing enables anoperator, one who may be observing directly or via remote telemetry, tomake real time corrections as the print develops.

A quiescent, biologically compatible environment may be provided duringone or more steps of the biomanufacturing process. One or morecomponents of the biomanufacturing facility 1 can control both thetemperature and humidity environment. Typical spacecraft ambientenvironments are in the temperature range of 20-25° C. with a lowrelative humidity in the range of 30-50%. The enclosable internal volumeof biomanufacturing facility 1 surrounding 3D bioprinter 2 can bemaintained and controlled at approximately the same ambient temperaturein the range of 20-25° C., but relative humidity may be controlled at anelevated but noncondensing 70-90% in order to mitigate desiccation ofthe printed structure while it is being processed. Atmospheric controlsystems used to heat, cool, humidify, and dehumidify enclosed volumes ina habitable reduced gravity environment are well known in the art ofclosed environmental life support systems used in spacecraft design.Some components of 3D bioprinter 2, specifically, print stage 12 andprint heads 17, may have active thermal control independent of the bulkinternal volume environment to enhance the quality and integrity of thebiomaterial being printed. If desired to be used, certain chemical orbiochemical reactions of the bioinks utilize heating or cooling relativeto the ambient environment of the 3D bioprinter at the time of extrusionor incorporation into the printed structure. The bioreactor encloses the3D printed structure and bathes it in liquid media thermally controlledto maintain the body temperature of the organism compatible with theprinted tissue. Typically, this will be human body temperature ofapproximately 37° C. The facility is designed with vibration isolationfor the print stage to insure the material is printed with highprecision.

After the tissue is printed, whether in the present bioprinter 2 oranother, these neo-tissues need to mature before they gain the strengthand function to be transplantable. This maturation will be accomplishedin a bioreactor 3 that utilizes a feedback control system to supply thegrowing tissue with oxygen and nutrient medium (glucose, trace elements,etc.), remove waste metabolic products and carbon dioxide, maintainoptimal pH, minimize the accumulation of air bubbles, and facilitatecell proliferation, differentiation, and tissue remodeling. The designof compatible bioreactors is well known in the art including bioreactorsdesigned for use in low gravity (such as in U.S. Pat. No. 7,198,940incorporated herein by reference). Typically they include means (notshown) for a liquid media supply reservoir, a liquid waste reservoir,pumps for circulating media, heating and cooling, oxygenation,degasification, sensors, and monitored feedback control systems.

In one embodiment, the printed structure may be physically removed fromprint stage 12 and transferred into a separate cell culturing bioreactor3 by an operator or robot. In this embodiment, bioreactor 3 may becollocated along with bioprinter 2 within biomanufacturing facility 1.Alternatively, bioreactor 3 may be in a different location notassociated with biomanufacturing facility 1. A removable bioreactor 3may be packaged to include its own integrated life support systems sothat it can serve a second function of maintaining the printed andcultured tissue in fluid living homeostasis for transportation andreturn to a patient on Earth or an alternate, extraterrestrialdestination. Such a ‘transportation compatible bioreactor’ would includeadequate robustness of design to survive the vibration environmentexperienced during planetary descent and possible refrigerationnecessary to extend longevity of the printed and cultured tissue. In analternate embodiment, a removable bioreactor 3 may be designed tointerface with a host transportation carrier capable of providing powerand life support to at least one bioreactor during transportation andreturn.

In a second embodiment, the print stage itself may be transformed into acell culture type vessel that becomes bioreactor 3. This may beaccomplished by simultaneously or serially printing both a bioink and athermoplastic using two print heads to create both the biologicalstructure and its enclosing culture vessel.

In a third embodiment, bioreactor 3 may comprise an open-toppedprefabricated and pre-plumbed enclosure 20 mounted on print stage 12.This third embodiment allows print head 17 the requisite access to printstage 12 to dispense the bioinks within pre-plumbed enclosure 20. Oncethe bioprinted structure is completed, either a prefabricated lid may beinstalled and sealed on enclosure 20 to create bioreactor 3 or,alternatively, a second print head 17 could extrude thermoplastic toprint a lid or top fastener directly on prefabricated enclosure 20.

Any of these embodiments may be enhanced by bioreactor 3 providing anyof equibiaxial mechanical loading in tension, electrical stimulation,fluid shear, or compression. Mechanical loading in tension may beprovided by printing the tissue construct on a flexible membrane. On theopposite side of the flexible membrane, a pressure or vacuum source maybe attached causing the membrane to distend and impart tension into thetissue. This stretching is known to induce maturation in many cell typesincluding cardiomyocytes. The bioreactor may include means to bothinduce and monitor electrical stimulation for depolarization currents incardiac tissues. The ability to capture the spontaneous contraction ofcardiomyocytes and pace a tissue is another indication of maturation.Terrestrial bioreactors providing mechanical loading, electricalstimulation, fluid shear, or compression are known in the art.

One embodiment of the reduced gravity biomanufacturing system having thepreviously described hardware can have at least one suite of softwaretools to create, edit, import, model, simulate, and control thebiomanufacturing system to produce the tissue and the supportingstructures or components for the creation, culture, transfer, orimplantation of the printed tissue. The system can import and modifyimage files from medical imaging formats to create geometries definingtissues or organs to be printed. The imaging technology can be selectedfrom magnetic resonance imaging, computerized tomography, X-rayradiography, medical ultrasound, endoscopy, tactile imaging, medicalphotography, positron emission tomography, and nuclear magneticresonance imaging. The system may output an electronic model file usedby another software or hardware platform to visualize the tissue before3D printing. The biomanufacturing system software tools can modify thetissue model in either two-dimensional sketching or three-dimensionalmodeling environments to correct, clarify, add, modify, remove orgenerally change the imported or originally produced geometry. Themethod can be done with bound or unbound constraints and can be drivenby individual changes, a lookup table or a mathematical equation boundby user defined variables.

Some biomanufactured tissues or organs may include prefabricatedstructures such as large blood vessels that are incorporated before,during, or after 3D bioprinting in reduced gravity. Prefabricatedstructure of the tissue or organ can be created by at least one ofelectrospinning, electrospraying, electroaerosoling, orelectrosputtering and can have three-dimensional scaffolds within orupon the bioprinted tissue or organ (See FIG. 6 prior artelectrospinning hardware). This method can be used for at least one ofthe creation of the tissue or organ, support structure, perfusion aid,implantation aid, cell delivery, or reagent delivery. Additionally,prefabricated sensors can be incorporated into the 3D printedbiomaterial or printed in place using electrically conductivebiocompatible feed stock. These incorporated sensors may have theability to provide data during the maturation process or followingimplantation in a target organism such as a human patient.

The reduced gravity biomanufacturing system may include a materialdispensing system capable of printing bioinks. One method ofmanufacturing includes wherein part or all of the tissue may be createdusing a print head utilizing a direct-write printing approach driven bymechanical plunger driven by vacuum, pressurized gas, pressurized fluid,linear motor, or drive screw to express the bioink. The dispenser tipsfor the print head can have a single or multiple bores to express one ormore bioinks simultaneously. The dispenser tips can be driven by asingle or multiple print heads. The system can contain one singlematerial or multiple materials. The method wherein part or all of thestructure of the tissue or organ can be created by at least one ofelectrospinning, electrospraying, electroaerosoling, orelectrosputtering. The methods can incorporate three-dimensionalscaffolds within or upon the bioprinted tissue or organ. The methods canbe used for at least one of the creation of the tissue or organ, supportstructure, perfusion aid, implantation aid, cell delivery, or reagentdelivery. The reduced gravity biomanufacturing system may print anaccurate, biologically viable reproduction of the desired tissue ororgan and can transfer the printed tissue or organ into a perfusedbioreactor chamber upon completion of printing automatically.Alternatively, the transfer may be done manually. The process can beperformed robotically with reduced damage or contamination of the tissueor organ printed. The reduced gravity biomanufacturing system mayculture the printed tissue or organ automatically to mature the tissue.The system may provide a method to perfuse the tissue and the developingvascular network. The system may allow the tissue to be removed from thereduced gravity biomanufacturing system and returned to Earth whileremaining viable and suitable for transplant. The reduced gravitybiomanufacturing system can be cleaned in place and reset withexpendable bioreactor chamber, print head cartridges, dispenser tips,media, bioinks and image file by an astronaut and verified remotely.

Bioreactor

In some implementations as shown in FIGS. 10-17 , the one or morebioreactors 300 may be constructed for receiving one or more tissues 150(e.g. same or different tissues) corresponding to the requirementsneeded to print and/or culture the desired tissue. The bioreactor 300may be used to condition or culture a number of tissues 150, includingskeletal muscle, bone, cartilage, neuronal, kidney, liver, etc. In someembodiments, the bioreactor 300 may include an ADSEP (AdvancedSeparation by Phase Partitioning) cassette, or a MVP (MultipurposeVariable-g Platform) cassette permitting culturing constructs invariable gravity environments (e.g. microgravity to 2 g). The bioreactormay provide post-print conditioning of the 3D bioprinted constructs andtissues 150 by construct perfusion and/or imparting cues to drivedifferentiation of the construct. The bioreactor 300 may be used toprovide at least one of perfusion, electrical stimulation, and/ormechanical stimulation to the construct 150. Once the constructdimensions pass the diffusion distance of gasses, ions, metabolites, andproteins, an internal network of printed blood vessels (e.g. vessel-likestructures) may deliver these entities to the internal cells of the 3Dprinted construct. With perfusion established in the bioreactor, thecells may be given cues to drive them to differentiate. For example, inapplications where the printed tissue is cardiac tissue, a variety ofmechanical and/or electrical stimulations may provide a conditioningenvironment similar to signals within or from a heart.

In some implementations, the bioreactor 300 may include a housing 310for conditioning the printed construct 150. The housing 310 may define avolume 311 therein. The housing 310 may include a lid 312 and base 313positionable between an open position (see FIG. 10 ) and a closedposition (see FIGS. 11 and 12 ). When in the open position, the tissuemay be printed into the base 313 and/or on one or more print platforms314. Alternatively, the printed tissue 150 may be transferred to thehousing 310 in some embodiments. When in the closed position, the lid312 may be sealed with the base 313. For example, one or more O-rings orseals (not shown) may be used between the lid 312 and base 313 to createa sealed volume 311 therein. In some embodiments, a portion of thehousing 310 (e.g. lid) may be translucent or transparent allowing visualconditions of the printed tissue to be observed while conditioning. Forexample, the bioreactor 300 may include a camera, video system, sensors,or monitoring device 301 to visually observe or other monitoring systemsto track conditioning. Monitoring the conditions or characteristics ofthe tissue (e.g. visual) may allow for adjustments (e.g. manual and/orautomatic) of one or more materials/media via the perfusions, electricalstimulation, mechanical stimulations, and/or other cues during theconditioning.

In some embodiments, the housing 310 may include a locking mechanism 302to secure the lid 312 in the closed and/or sealed position with the base313. As shown in FIG. 10 , the one embodiment of the locking mechanism302 is one or more clamps or fasteners sealing and/or locking the lid312 with the base 313. Locking the lid may also compress the one or moreseals/gaskets creating the seal for the internal volume 311.

In some implementations, the bioreactor may provide one or moreperfusions to the printed construct 150. As shown in the figures, thebioreactor 300 may include one or more perfusion ports 331 adapted to bein fluid communication with the 3D printed construct 150. The perfusionports 331 may be in fluid communication with the printed tissue 150 inone directional flow (see FIG. 17 ) as shown in the one embodiment,however the fluid flow may be in more than one direction. The one ormore fluid flows through the one or more ports 331 may be a variety offlow rates (e.g. variable or constant). For example, one or more ports331 may have the same or different flow rates. Moreover, the flow ratesmay change or be different within a single port or array of ports. Theone or more ports 331 (e.g. outlet ports) may also be positioned indownstream fluid communication with the 3D printed construct 150 and theupstream ports 331 (e.g. intake ports) may be positioned upstream of theconstruct 150. The one or more ports 331 (e.g. intake and/or outletports) may be a variety of quantities, positions, constructions, shapes,and sizes and still be in communication with the 3D printed tissues(e.g. vessel structure).

In some implementations, the bioreactor 300 may include one or moremanifolds or members 330 engaging the constructs 150. The one or moremanifolds 330 may be in fluid communication or perfusion with the 3Dprinted construct 150. One or more intake manifolds 330 a may includeone or more ports 331 (e.g. intake ports). One or more outlet manifolds330 b may include one or more ports 331 (e.g. outlet ports). The intakemanifold 330 a and outlet manifold 330 b (e.g. ports) may be in fluidcommunication with the 3D printed construct. In some embodiments, theintake manifold 330 a may include a plurality of channels 332 incommunication with one or more of the ports 331 therein. The channels332 may provide for substantially the same flow rate or material to passthrough the one or more ports 331 of the intake manifold 330 a. The oneof more channels 332 or ports 331 of the intake manifold 330 a may besupplied by at least one supply conduit 333. As shown in the oneembodiment in FIGS. 15 and 16 , the outlet manifold 330 b may includedownstream engagement with the construct 150 (e.g. vessel structures).The ports 331 of the outlet manifold 330 b may be in communication withthe volume 311 of the housing 310 via an interconnecting chamber 334within the outlet manifold 330 b. If a chamber 334 is used, the chambermay be recessed within the outer periphery of the manifold.

In some embodiments as shown more clearly in FIGS. 13-16 , the one ormore ports 331 may be defined by one or more needles 335 in one or moreof the manifolds. As shown in the figures, both the intake and theoutlet manifolds 330 a, 330 b have ports 331 defined by needles 335projecting inwardly towards the printed construct 150, print platform314, and/or other manifold 330. The ports 331 and/or needles 335 may beinserted into structure of the 3D printed construct 150. The ports 331and/or needles 335 may be a variety of shapes, sizes, quantities,constructions, and/or have a variety of through openings therein. Forexample, the ports 331 may have uniform/non-uniform inner diameters.

The manifold and/or ports may be subsequently engaged to the printedconstruct 150 after printing with the 3D bioprinter 2. Alternatively,the printed structure may be printed on one or more portions of one ormore manifolds 330. One or more manifolds 330 (e.g. intake and/or outletmanifolds) may be positioned out of engagement with the 3D printedconstruct 150 and subsequently engaged with the 3D printed construct.Further, the manifolds 330 may be movable, positioned, or engaged withthe printed construct manually and/or automatically by a drive mechanismor system. As shown in FIG. 11 , the printed construct 150 may beprinted within the bioreactor 300 (e.g. print platform 314 or volume311) spaced away from one or more manifolds 330 in a disengagedposition. As shown in FIG. 17 , the manifolds 330 are positioned to anengaged position with the construct 150. One embodiment of a drivemechanism 341 (e.g. motor, belt, solenoid, etc.) that may be used tomove (e.g. translate) the manifolds between the engaged and disengagedposition with the construct 150 is shown in the figures. This drivemechanism 341 moves both manifolds into engagement with the 3D constructafter printing. It should be understood that one manifold may be movedinto engagement in some embodiments instead of a plurality of manifolds.The bioreactor 100 may include attachment mechanisms to secure themanifolds with the construct (e.g. during conditioning). The one or moremanifolds or portions thereof (e.g. needles or ports) may include theattachment mechanism (not shown) such as, but is not limited to,chemical (e.g. adhesive) and/or mechanical attachments to the 3D printedconstruct. For example, an adhesive (e.g. bio adhesive) may be appliedto or be present on the manifold or portions thereof to attach to theprinted construct. Alternatively or in combination with the attachmentmechanism of the bioreactor, the construct may include the attachmentmechanism or portions thereof. For example, the printed tissue ormaterial(s) thereof may include a bio adhesive to secure the interfaceof the manifold/ports and the 3D printed tissue. These attachmentmechanisms may allow or assist in maintaining connectivity/communication(e.g. mechanical, electrical, and/or fluid communication) with theconstruct during mechanical manipulation or conditioning within thebioreactor (e.g. forces applied to the construct via one or moremechanical stimulation devices 340)

In some implementations, the bioreactor 300 may have one or moremechanical stimulation devices 340. The mechanical stimulation device340 may be a variety of mechanisms or constructions manipulating theprinted construct. The mechanical stimulation device may apply a varietyof forces to the 3D construct in a variety of directions relative to theconstruct. As shown in the one embodiment, the mechanical stimulationdevice 340 may include a drive mechanism 341 embodiment having a motor342 (e.g. stepper motor), drive belt 343, and a lead screw 344manipulating the construct 150 in one or more directions. The drivemechanism 341 may move one or more of the manifolds 330 (e.g. intakeand/or outlet manifold) at the same and/or differentrates/times/durations. In the one embodiment shown, the drive mechanism341 drives both manifolds 330 a, 330 b. In the one embodiment shown, thedrive mechanism 341 may compress and/or extend the 3D printed construct150 during conditioning with the manifolds. It should be understood thatthe same or different drive mechanism 341 that engages/disengages fromthe construct may apply the mechanical stimulation. As shown in the oneembodiment, the drive mechanism 341 does both. The drive mechanism 341applying the desired mechanical manipulation of the construct may also,in some embodiments, be configured to disengage from the 3D constructand/or engage with the 3D construct. The drive mechanism 341, in the oneembodiment shown, drives the intake manifold 330 a and the outletmanifold 330 b towards each other (e.g. compression or compressedposition) or away from each other (e.g. extending/extension or extendedposition) along a linear path or axis. Further, the manifolds 330 moveat the same rate in the one embodiment shown. It should be understoodthat the manifolds may move at different rates, times, duration, and/ordirections. For example, the mechanical stimulation device 340 (e.g. oneor more of the manifolds) may apply compression, extending, and/orrotationally forces to the 3D construct in some embodiments. The drivemechanism or mechanical stimulation devices 340 may include a variety ofseals 345 for sealingly engaging portions thereof (e.g. lead screwand/or guide pins 344) with the housing 310, volume 311, or otherportions of the bioreactor.

In some implementations, the bioreactor 300 may have one or moreelectrical stimulation devices 350. The electrical stimulation device350 may be a variety of constructions, quantities, shapes, sizes, andpositions and still be in electrical communication with the 3D construct150. In the one embodiment shown as shown more clearly in FIGS. 13-16 ,the one or more manifolds 330 may include one or more electricalstimulation devices 350. The electrical stimulation device 350 mayinclude one or more conductive materials or members 351 engaging the 3Dconstruct 150 to transfer electrical stimulation (e.g. voltage) throughor into one of more portions of the construct. The electricalstimulation may be a variety of values, rates, sequences, durations, andengagement positions (e.g. contact surface area, penetration, etc.) withthe construct to condition the corresponding tissue. In the oneembodiment shown, each one of the intake manifold 330 a and the outletmanifold 330 b includes the conductive material 351 of the electricalstimulation device 350. Therefore the electrical stimulation may passfrom the intake manifold 330 a through the printed tissue 150 andcontinue to the outlet manifold 330 b. One or more surfaces of themanifold 330 may include the conductive material 351. When the one ormore manifolds 330 engage the 3D construct, the conductive material 351may also engage the 3D construct. The manifolds 330 may be constructedof a variety of materials, although a ceramic may be used. Theconductive material 351 may be an outer cover, exterior coating, orcontact layer overlying the manifold (e.g. ceramic material) to contactthe 3D construct. In the embodiment shown, the conductive material 351may include a planar portion 352 and/or needles 353 (e.g. solid)projecting away from the manifold 330. The needles 353 may extend fromthe planar portions 352. These needles 353 may not define one or moreports. These needles or other structure may engage or penetrate the 3Dconstruct at one or more depths, while the planar portion or otherstructure of the conductive material may engage the outer periphery ofthe 3D construct.

In some implementations, the bioreactor 300 may include one or morefluid traps 360 (e.g. trapping air or bubbles). The one or more air orfluid traps 360 may be in fluid communication with one or more of thefluid communications or conduits with or within the bioreactor 300 orhousing 310. This removal of air may be in fluid flowrecirculated/circulated, introduced to the bioreactor/printed tissue,and/or removed from the bioreactor or tissues located therein. The airtraps 360 may reduce the air or bubbles generated during culturing,within the system/bioreactor, or circulated with other substances ormedia. The fluid or fluid pathways may be moved by one or more pumps 361through and/or around the 3D printed construct. One or more valves 362(e.g. a three way valve) and operating controls 363 may be used tocirculate or move fluid relative to the bioreactor.

In some implementations, the bioreactor may include one or morefeed/media bags 364 and/or pumps 361 in fluid communication with theconstruct 150, the housing 310, or portions of the bioreactor 300. Asshown in FIG. 17 , the one or more media bags 364 may supply fluid/mediaor be in fluid communication with the volume 311 of the housing 310.Moreover, the bioreactor 300 may include one or more valves 362 orcontrols 363 operating the flow with the media bag 364 and/or air trap360. The air trap 360 may be in fluid communication with one or moremedia bags 364 and at least one port 315 of the housing 310. The mediabags 364 may be detachable with the bioreactor for replacement withanother media bag with similar media content or different media content.

In some implementations, the bioreactor 300 may include one or morewaste bags 365 and/or pumps 361 in fluid communication with theconstruct 150, housing 310, or portions of the bioreactor 300. As shownin FIG. 17 , the one or more waste bags 365 may be in fluidcommunication with the volume 311 of the housing 310. Moreover, thebioreactor may include one or more valves 362 or controls 363 operatingthe fluid flow with the volume 311 of the housing 310 or portions of thebioreactor 300. The waste bag 365 may be in downstream fluidcommunication with one or more ports 315 of the housing 310. The wastebags 365 may be detachable with the bioreactor for replacement withanother waste bag.

In the embodiment shown, the bioreactor 300 may include one or more airtraps 360 in fluid communication with one or more ports 331 engaging the3D printed construct 150, media bags, manifolds 330 (e.g. intakemanifold), and/or other portions of the bioreactor 300. In someembodiments as shown in FIG. 17 , one or more pumps 361 may be used tocirculate media/substrate or fluid through one or more manifolds 330and/or the printed tissue 150. Moreover, the circulated fluid or mediamay be pumped into, through, and/or from the volume 311 of the housing310. As shown in the one embodiment in FIGS. 13, 14, 14 a, and 17, therecirculated fluid path may extend at least from the air trap 360through the port 315 of the housing 310 and divides from the supplyconduit 333 into the one or more channels 332 of the intake manifold 330a to exit through the intake ports 331. As further shown in FIG. 17 ,upon passing from the intake manifold ports 331, the fluid may passthrough the 3D printed construct 150 (e.g. vessel structure) beforeexiting through the ports 331 and/or outlet manifold 330 b (e.g. chamber334) and into the volume 311 of the housing 310. The fluid within thehousing 310 may then exit through another port 315 of the housing 310and continue back to the air trap 360 via the pump 361. Moreover, thebioreactor may include one or more valves 362, pumps 361, or controls363 operating the flow with the 3D printed construct 150, manifolds 330,volume 311, and/or ports 331.

In some implementation, the bioreactor 300 or portions thereof may beprimed with media or desired fluid. Priming one or more portions of thebioreactor may reduce the air circulated and/or remove air from one ormore portions of the bioreactor. In the one embodiment shown in FIG. 17, the volume 311 defined by the housing 310 may be primed manually witha fluid or media (e.g. saline or desired media) manually with a syringe366 and/or an air waste bag 367 attached thereto. Alternatively, a mediabag 364 may be attached to the housing and pumped therein to fill up thevolume 311. Moreover, in some embodiments as shown in FIG. 17 , thesupply conduit 333 to the intake manifold 330 a may be primed with asyringe 366 with a fluid or media (e.g. saline or other desired media)via an input with the recirculation pathway. The priming through thesupply conduit 333, may remove air or prime the manifold (e.g. ports orintake manifold). For example, the intake manifold may be primed beforeengaging the construct 150 to reduce or eliminate air delivered to theconstruct.

In the embodiment shown, the lid 312 and base 313 may also have one ormore interior surfaces defining the volume 311 therein constructed toreduce the trapping of air and/or increase the removal of air. Theinterior surfaces defining the inner periphery of the housing 310 orvolume 311 may be rounded with large radii and/or have chamfered edges.The chamfered or rounded edges may be used on one or more portions orsurfaces within the bioreactor (e.g. lid, base, manifolds, printplatform, etc.).

Although not shown, the bioreactor 300 may include one or more lightsources. The light sources may illuminate the contents or volume of thebioreactor or portions of the bioreactor to aid in visual monitoring(e.g. monitoring device 301).

In use, the bioreactor 300 may include a cassette or cassette housing316. The cassette 316 may include a sealable lid 317 as shown in FIG. 10. The cassette 316 and/or bioreactor 300 may be engaged with the 3Dbioprinter 2 and/or biomanufacturing facility 1 within the reducedgravity environment 30 with the housing lid 312 and/or cassette 316(e.g. lid 317) opened. One or more 3D constructs 150 may be printed intothe one or more bioreactors 300 and/or within the opened housing 310.For example, the printed 3D construct may be printed adjacent to or onthe print platform 314. The intake manifold 330 a and/or ports 331 maybe primed or air is removed from one or more fluid lines or conduits.This priming or evacuation of air from the intake manifold 330 a orports 331 may be from a bypass (e.g. via a syringe 366) and/or bypumping media or substrate (e.g. saline) through the recirculationlines/pump. The manifold 330 and/or ports 331 of one or both of theintake/outlet manifolds 330 a, 330 b may be in the disengaged positionfrom the printed tissue 150 and moved towards and engages the printedtissue in the engaged position. The perfusion ports 331 (e.g. needles),electrical stimulation device 350 (e.g. needles and/or planar portions),and/or mechanical stimulation devices 340 may engage the printed tissuein at least one position (e.g. engaged position) of the one or moremanifolds. Attachment mechanisms (e.g. adhesive and/or mechanicalattachments) may engage one or more portions of the manifolds 330 withone or more portions of the 3D construct 150. For example, mechanicaland/or chemical attachments (e.g. adhesives) may engage the ports 331with vessel structure of the printed tissue 150. Further, theelectrical/mechanical stimulation devices may be engaged bymechanical/chemical attachments. The lid 312 of the housing 310 may beclosed and/or sealed with one or more locking mechanisms 302 (e.g.clamps). One or more media bags 364 may be fluidly attached the housing310 or portions of the bioreactor 300. One or more media waste bags 365may be fluidly attached to the housing 310 or portions of the bioreactor300. One or more air waste bags 367 may be fluidly attached to thehousing 310 or portions of the bioreactor 300. With the volume 311 ofthe housing 310 sealed, the air within the volume may be evacuated orremoved to the air waste bags 367. The air may be manually removed via asyringe 366 by introducing saline or media into the housing 310 to pushair out of the volume 311 and into the air waste bag 367. Alternatively,and or in combination with the manual evacuation, the air from thevolume 311 may be pushed out of the housing 310 by pumping media in fromone or more media bags 364. The cassette 316 containing the bioreactorhousing 310 may be closed/sealed. At some point during use, the cassette316 or bioreactor 300 may be removed from the 3D bioprinter 2.

In use, the 3D printed construct 150 may be conditioned or culturedwithin the reduced gravity environment 30. The recirculation of media,addition/removal of media, perfusion stimulation, collection and/orremoval of waste, and/or removal of air/bubbles, the electricalstimulation, and/or mechanical stimulation within the one or morebioreactors may be operated by one or more operating controls 363.Alternatively, or in combination thereof, one or more of these steps oroperations may be completed manually. Air may be removed from thecirculation of media or fluid flow within the bioreactor or to themanifolds 330/ports 331. Air may be removed from media introduced fromthe media bag 364 to the housing 310. When needed, waste may be removedfrom the housing. Additional media or supplemental media may be added tothe bioreactor 300 at various stages or conditions of the 3D construct.The 3D construct may have one or more perfusion ports providing mediathereto. One or more electrical stimulations may be provided to the 3Dconstruct. One or more mechanical stimulations (e.g. compression,extending, and/or rotational forces) may be provided during theconditioning of the 3D construct. The bioreactor may monitor the 3Dconstruct and/or provide the perfusions and/or electrical/mechanicalstimulations when needed for conditioning. Once a bioreactor and/orcassette is removed from the 3D printer, another bioreactor may beengaged or positioned with the 3D bioprinter and begin printing another3D tissue (e.g. the same or different tissue).

While several embodiments have been described and illustrated herein,those of ordinary skill in the art will readily envision a variety ofother means and/or structures for performing the function and/orobtaining the results and/or one or more of the advantages describedherein, and each of such variations and/or modifications is deemed to bewithin the scope of the embodiments described herein. More generally,those skilled in the art will readily appreciate that all parameters,dimensions, materials, and configurations described herein are meant tobe exemplary and that the actual parameters, dimensions, materials,and/or configurations will depend upon the specific application orapplications for which the teachings is/are used. Those skilled in theart will recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, embodiments may bepracticed otherwise than as specifically described and claimed.Embodiments of the present disclosure are directed to each individualfeature, system, article, material, and/or method described herein. Inaddition, any combination of two or more such features, systems,articles, materials, and/or methods, if such features, systems,articles, materials, and/or methods are not mutually inconsistent, isincluded within the scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

It is to be understood that the embodiments are not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the description or illustrated in the drawings.The invention is capable of other embodiments and of being practiced orof being carried out in various ways. Unless limited otherwise, theterms “connected,” “coupled,” “in communication with,” and “mounted,”and variations thereof herein are used broadly and encompass direct andindirect connections, couplings, and mountings. In addition, the terms“connected” and “coupled” and variations thereof are not restricted tophysical or mechanical connections or couplings.

The foregoing description of several embodiments of the invention hasbeen presented for purposes of illustration. It is not intended to beexhaustive or to limit the invention to the precise steps and/or formsdisclosed, and obviously many modifications and variations are possiblein light of the above teaching.

What is claimed is:
 1. A bioreactor for receiving a printed tissuecomprising: a housing having one or more manifolds therein adapted toengage a printed tissue; the one or more manifolds having each one of aplurality of perfusion ports in fluid communication with a printedtissue; one or more electrical stimulation devices; and a mechanicalstimulation device connected thereto, wherein at least one of the one ormore manifolds contains the plurality of perfusion ports defined by oneor more needles; and wherein the one or more electrical stimulationdevices is an exterior coating to the one or more manifolds.
 2. Thebioreactor of claim 1 further comprising a print platform.
 3. Thebioreactor of claim 1 wherein the mechanical stimulation device movesthe one or more manifolds between a compressed position and an extendedposition.
 4. The bioreactor of claim 1 wherein the exterior coatingincludes one or more needles.
 5. The bioreactor of claim 1 wherein theone or more manifolds include an intake manifold and an outlet manifold.6. The bioreactor of claim 5 wherein at least one of the intake manifoldand the outlet manifold includes one or more needles defining theplurality of perfusion ports.
 7. The bioreactor of claim 5 wherein atleast one of the intake manifold and the outlet manifold includes one ormore channels in communication with the plurality of perfusion ports. 8.The bioreactor of claim 5 wherein at least one of the intake manifoldand the outlet manifold are moveable relative to each other.
 9. Thebioreactor of claim 1 wherein the one or more electrical stimulationdevices include a conductive material positioned on one or more surfacesof the one or more manifolds.
 10. The bioreactor of claim 9 wherein theconductive material includes one or more needles projecting and adaptedto engage a printed tissue.
 11. The bioreactor of claim 1 wherein themechanical stimulation device includes a drive mechanism moving at leastone of the one or more manifolds.
 12. The bioreactor of claim 1 furthercomprising one or more air traps in fluid communication with theplurality of perfusion ports.
 13. The bioreactor of claim 1 furthercomprising one or more detachable feed bags and waste bags.